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How to determine the technical parameters for method validation: 1. Prerequisites for successful method validation: (1) The instrument has been verified, calibrated, and is within its valid period; (2) Trained personnel; (3) Reliable and stable reference materials and standard samples; (4) Reliable and stable reagents for experiments; (5) It must be confirmed that the test solution remains stable and does not degrade over the specified time period. 2. Accuracy: Accuracy refers to the degree to which the results obtained using this method are close to the true value or reference value, and it is expressed as a percentage recovery rate. The specific method for determining the recovery rate R is the spiked recovery test. The spiking recovery test determines the content of P in the sample by measuring a genuine sample plus a control (or standard) containing a known amount A, with the measured value being M. Data requirements: Within the specified range, evaluation should be based on at least 9 measurement results; for example, three samples at high, medium, and low concentrations should each be measured three times to assess precision (repeatability, intermediate precision, and reproducibility refer to the degree of consistency among the results obtained from multiple measurements of the same uniform sample under specified conditions). It is expressed in terms of deviation (d), standard deviation (SD), and relative standard deviation (RSD) (coefficient of variation, CV). (1) Repeatability: The precision of the results obtained by the same analyst under identical conditions ； Within the specified range, the evaluation should be based on at least 9 measurement results; for example, three samples at different concentrations can be measured three times each, or if the concentration of the substance being tested is set at 100%, at least 6 measurements are required for evaluation. (2) Intermediate precision: refers to the precision of the results obtained by different analysts using different equipment at different times within the same laboratory. (3) Reproducibility: refers to the precision of the results obtained by different analysts in different laboratories. (4) Data requirements: SD, RSD, and confidence intervals must be reported. Specificity refers to the ability of a method to accurately determine the characteristics of the substance being tested even in the presence of other components such as impurities, degradation products, or excipients. It indicates whether the method can accurately and specifically identify the test substance when co-existing substances are present, and it serves as a measure of the degree of interference when using this method for analyzing complex samples. Typically, this is determined by analyzing samples containing impurities, degradation products, related chemical substances, or placebo components, and comparing the results obtained with those from samples without these components; the difference between the two sets of results reflects the specificity. A specific reaction should be able to distinguish between the substance being analyzed and potentially co-existing substances or compounds with similar structures; samples that do not contain the substance being analyzed, as well as compounds with similar structures or components, should yield a negative reaction. For quantification of the substance’s content and detection of impurities, chromatographic methods and other techniques should be accompanied by representative chromatograms that also demonstrate specificity ； The positions of each component should be indicated in the diagram, and the resolution in chromatography must meet the requirements. • When impurities are available, for assay purposes, impurities or excipients may be added to the sample to examine whether the test results are affected ； For the determination of impurities, a certain amount of impurities can also be added to the sample to examine whether they can be separated from one another. 5 Detection limit: The detection limit refers to the lowest concentration or amount of the analyte that can be detected in a sample under specified experimental conditions ； It is an indicator of the efficiency of limit testing; no quantitative measurement is required – it is sufficient to indicate whether the concentration is above or below the specified value. • Visual method in instrumental analysis: A substance of known concentration is used to determine the lowest concentration or amount that can be detected reliably. • Signal-to-noise ratio method: Used in analytical methods that allow for the detection of baseline noise (instrumental analysis methods); it involves comparing the signal obtained from a sample with a known low concentration with the signal obtained from a blank sample, in order to determine the lowest concentration or amount that can be detected reliably ； The detection limit is generally determined based on the corresponding concentration or the amount injected into the instrument at a signal-to-noise ratio of 3:1 or 2:1. The standard deviation method can also be used: with a blank value of 0 ； ①Measure the background more than 10 times and calculate the standard deviation σ ； ②Multiply σ by three ； ③The concentration X corresponding to 3σ on the working curve is the detection limit of the method. When the blank value is not equal to 0, ① measure the background more than 10 times to determine the standard deviation σ ； ②Multiply σ by three ； ③Find the concentration X corresponding to 3σ on the working curve ； ④The obtained corresponding concentration value plus the blank value gives the detection limit of this method. 6 Quantitation limit • It refers to the lowest amount of the analyte in a sample that can be quantitatively determined; the results must meet certain accuracy and precision requirements. • The signal-to-noise ratio method is commonly used to determine the quantitation limit. Generally, it is defined as the concentration or amount injected into the instrument at which the signal-to-noise ratio (S/N) is 10:1. Alternatively, 10 times the standard deviation (SD) of the blank background response measured by the instrument can be used as an estimate, with the actual lower limit of quantification being determined through experiments. 7 Linearity • It indicates the degree to which test results are directly proportional to the concentration of the analyte in the sample within the designated range. The linearity is usually represented by the slope of the regression curve, which is obtained by using the least squares method to process the data. Data requirements: At least five concentrations are needed to examine linearity; parameters such as the correlation coefficient, y-intercept (which represents the possible deviation in testing), regression slope, and variance must be provided. The number of regression equations and the linear plots should also be included. Note: When performing linearity testing, it is advisable to use as many data points as possible and ensure a wide linear concentration range. The advantage of this is that when evaluating accuracy, the concentration range for recovery testing will be broader, making it easier to prepare the solutions ； Otherwise, when conducting a linearity test, if the range of linear concentrations you use is not wide enough, then when evaluating the recovery rate using the spike addition method, the range of concentration changes will also be limited. In such cases, it becomes difficult to control the operating concentration, leading to difficulties in actual operations. The range refers to the interval of upper and lower concentration or quantity limits within which a test method is applicable, under conditions where a certain level of precision, accuracy, and linearity is achieved. The range should be determined based on the specific application of the analytical method, as well as the results and requirements regarding linearity, accuracy, and precision. Taking pharmaceutical analysis as an example: • The acceptable range for the assay of active pharmaceutical ingredients and formulations is 80%–120% ； The uniformity of content in the preparation ranges from 70% to 130% ； The impurity determination should be at 120% of the reported value of the impurity in question up to its limit ； The dissolution rate should be within ±20% of the specified range; if a limit range is defined, it should be from -20% of the lower limit to +20% of the upper limit. For example, if the dissolution rate for sustained-release tablets is 70% after 1 hour, then the validation range is set at 0–90%. 9 Durability refers to the degree to which the results remain unaffected by slight changes in testing conditions, providing a basis for routine inspections. It is a measure of the reproducibility of experimental results between laboratories and researchers under normal conditions ； If the method is sensitive to analytical conditions or requires stringent conditions, this should be indicated. Typical changes include experimental conditions such as the stability of the analysis solution and extraction time.